Project Summary/Abstract The overarching theme of this research program is the utilization of a modular approach to synthesize highly-conjugated arylethynyl and P,N-heterocyclic receptors for anion recognition. Unlike the majority of fluorescent anion sensors, these sensors contain an inherently fluorescent backbone as opposed to a pendant fluorophore unit. The research will address fundamental problems in the development of the next generation of molecular probes and binding agents for anions and focuses on three main areas: (1) synthesizing and studying new recognition motifs and scaffolds to advance our modular design strategy; (2) quantifying the interactions of these receptors with anions, and (3) applying these compounds as new fluorescent molecular probes for chloride. The proposed receptors will also help provide a fundamental understanding of anion coordination chemistry. Anions are increasingly recognized as problematic environmental contaminants and are vital to many processes in nature, ranging from developing tertiary structure in proteins and DNA, to ?synergistic? binding of metals in proteins, to their increasingly understood role in biological processes including endocytosis, lysosome activity and kinase function, among others. Anion binding proteins and transport channels also may be involved in the mechanisms of a variety of disease pathways (e.g., Cystic Fibrosis, osteopetrosis, Alzheimer's disease, cancers, etc.). This research will provide fundamental insight into emerging new approaches to target anions (?anion-?-type? interactions, C-Hanion hydrogen bonding) and may provide novel selectivity in anion coordination. Understanding anion binding on a molecular level is of paramount importance if one wishes to elucidate the roles of anions in the much more complicated biological processes. The specific aims of the proposed research are: (1) to synthesize and study a series of receptors for anions that exhibit tunable OFF-ON fluorescence resulting from the modular design; (2) to elaborate a new class of fluorescent P,N-heterocycles as anion binding agents; and (3) to develop new water-soluble small molecule fluorescent probes for anions and to screen successful candidates in cell/lysate assays. We will investigate receptors developed on this project first using modern physical organic methods, including: molecular modeling for host design; host-guest titrations to quantify the energetics of binding interactions; linear free energy relationships and kinetic isotope effects to understand the fundamental nature of anion binding interactions; and a variety of spectroscopic techniques to assess solution speciation. Collaborations will enable screening of successful host molecules as molecular probes for in vitro and in vivo applications. In the long term, probes developed in this project can be used broadly by researchers studying anion homeostasis and ion transport.